Chen C.C. (ed.) Selected Topics in DNA Repair

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and Modulation of DNA Repair After Exposure to Gamma-Radiation

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suggested that the radioprotective efficacy by FA may be a result of early recovery of

hematopoietic cells due to enhanced production of G-CSF and erythropoietin (Ma et al.

2011a). They have also reported that FA had a radioprotective effect through the ERK

pathway to inhibit apoptosis and oxidation (Ma et al. 2011b).

5.6 Troxerutin

Troxerutin (Scheme 2D), a derivative of the natural flavonoid rutin extracted from Sophora

japonica (Japanese pogoda tree) has been commonly used in the treatment of Chronic

Venous Insufficiency (CVI) disease (Lefebvre and Lacombe 1991). In clinical trials,

troxerutin has been given in doses up to 7 g per day orally for up to 6 months with no

contraindications (Glacet-Bernard et al. 1994). It has been reported that during radiotherapy

of head and neck cancer, administration of a mixture of troxerutin and coumarin offered

protection to salivary glands and mucosa. We have shown that troxerutin inhibited lipid

peroxidation in membrane of sub-cellular organelles as well as normal tissues of tumor-

bearing mice exposed to gamma-radiation. Further, it was found that administration of

troxerutin resulted in differential protection of DNA in blood leucocytes and bone marrow

cells and not in cells of tumor in whole body irradiated tumor-bearing mice. Troxerutin

protected the human peripheral blood leucocytes from radiation-induced DNA strand

breaks in a concentration dependent manner under ex vivo condition of irradiation (2Gy)

Fig. 2. A. Baicalein, B. Chlorophyllin, C. Ferulic acid, D. Troxerutin, E. Vanillin.

Selected Topics in DNA Repair

492

(Maurya et al. 2004). Intraperitoneal administration of troxerutin (175 mg/kg body weight)

to mice before and after whole body radiation exposure inhibited micronuclei formation in

blood reticulocytes significantly. The administration of different doses (75, 125 and 175

mg/kg body weight) of troxerutin 1 h prior to 4 Gy gamma-radiation exposure showed

dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and

bone marrow cells. The dose-dependent protection was more pronounced in bone marrow

cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1

h prior or immediately after whole body irradiation of mice showed that the decrease in

strand breaks depended on the post-irradiation interval at which the analysis was done.

Measurement of DNA repair potential of the troxerutin shows that it enhances the DNA

repair (Maurya et al. 2005a). So the observed time-dependent decrease in the DNA strand

breaks could be attributed to enhanced DNA repair in troxerutin administered animals.

5.7 Vanillin

Vanillin (4-hydroxy-3-methoxybenzaldehyde), scheme 2E, is a compound used as a

flavoring agent and as a dietary component. It is the major component of natural vanilla,

which is one of the most widely used and important flavoring materials throughout the

world. The source of vanilla is the bean, or pod, of the tropical Vanilla orchid (principally

Vanilla planifolia Andrews, syn. V. fragrans (Salisb. Ames)). Vanillin is an antioxidant capable

of protecting membrane against lipid peroxidation and DNA against strand breaks induced

by reactive oxygen species like singlet oxygen. Vanillin and its analogs were strongly anti-

mutagenic or anti-genotoxic in most studies. Vanillin also suppresses the chromosomal

aberrations induced by X-rays in V79 cells in vitro (Keshava et al. 1998) and in mice in vivo

(Sasaki et al. 1990). Imanishi et al. have shown anti-mutagenic effect of vanillin against UV

and X-rays in Chinese hamster V79 cells (Imanishi et al. 1990). We have explored the effect

of vanillin on radiation-induced DNA damage in plasmid pBR322 (in vitro), human

peripheral blood leucocytes and mouse splenic lymphocytes (ex vivo) and in mouse (in vivo),

besides the possible mechanisms involved in terms of scavenging radiation related free

radicals by pulse radiolysis. Our finding shows that presence of 0.5mM vanillin has a dose-

modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human

peripheral blood leucocytes (ex vivo) to radiation causes strand breaks in the cellular DNA,

as assessed by comet assay. When leucocytes were exposed to 2 Gy of -radiation there was

an increase in parameters of comet assay such as %DNA in tail, tail length, ‘tail moment’

and ‘Olive tail moment’. The presence of 0.5 mM vanillin during irradiation significantly

reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after

whole-body exposure of mice (in vivo) to radiation was studied at 1 and 2 h post-irradiation.

There was recovery of DNA damage in terms of the above-mentioned parameters at 2h

post-irradiation. This was more than that observed at 1 h (Maurya et al. 2007). The recovery

was more in vanillin treated mice. Hence our studies showed that vanillin offers protection

to DNA against radiation-induced damage possibly imparting a role other than modulation

of DNA repair (Maurya et al. 2007).

6. Future prospects for radioprotectors in mitigation of radiation damage

Research in the development of radioprotectors worldwide has focused on screening a

variety of chemical and biological compounds (Maurya et al. 2006; Nair et al. 2001; Weiss

and Landauer 2009). Various natural or synthetic compounds having either antioxidant or

Role of Radioprotectors in the Inhibition of DNA Damage

and Modulation of DNA Repair After Exposure to Gamma-Radiation

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cytoprotective or immunomodulatory property or their combination have been evaluated

extensively for their radioprotective potentials in both in vitro and in vivo models (Maurya

et al. 2006; Weiss and Landauer 2009). So far no synthetic radioprotective molecule is

available that is perfectly safe and effective. Herbal origin radioprotectors have a hope to

overcome the toxicity issues seen with the synthetic compounds. There are many reports on

the pre-irradiation radioprotectors, using synthetic or natural compounds. There are hardly

a few post-irradiation radioprotectors. Such radioprotectors based on induction of DNA

repair and related mechanisms will have great potential in reducing accidental exposure

such as those that occur due to natural calamities like earthquakes and tsunami as in the

case of Fukushima daiichi nuclear reactors. To overcome this kind of situation, there is a

need to locate more effective post-irradiation effective radioprotector that can enhance the

DNA repair. Since the DNA is the critical target for ionizing radiation, future, studies

should focus on compounds that have potential to enhance the process of DNA repair

which is essential after radiation exposure. These also can be used in other cases of exposure

such as that of radiotherapy for cancer.

7. References

Charrier, S., Michaud, A., Badaoui, S., Giroux, S., Ezan E, Sainteny, F., Corvol, P., &

Vainchenker, W. (2004). Inhibition of angiotensin I-converting enzyme induces

radioprotection by preserving murine hematopoietic short-term reconstituting

cells. Blood Vol.104, No.4, pp. 978-985

Cherdyntseva, N., Shishkina, A., Butorin, I., Murase, H., Gervas, P., & Kagiya, T.V. (2005).

Effect of tocopherol-monoglucoside (TMG), a water-soluble glycosylated derivate

of vitamin E, on hematopoietic recovery in irradiated mice. Journal of Radiation

Research, Vol.46, No.1 pp.37-41

Chiu LC, Kong CK, and Ooi VE. (2003). Antiproliferative effect of chlorophyllin derived

from a traditional Chinese medicine Bombyx mori excreta on human breast cancer

MCF-7 cells. International Journal of Oncology, Vol. 23, No.3, pp.729-735

Damron, T.A., Spadaro, J.A., Horton, J.A., Margulies, B.S., Strauss, J.A., & Farnum, C.E.

(2004). Novel radioprotectant drugs for sparing radiation-induced damage to the

physis. International Journal of Radiation Biology, Vol. 80, No. 3, pp.217-228

Devasagayam, T.P., Kamat, J.P., Mohan, H., & Kesavan, P.C. (1996). Caffeine as an

antioxidant: inhibition of lipid peroxidation induced by reactive oxygen species.

Biochim Biophys Acta, Vol. 1282, No 1, pp.63-70

Diamond, A.M., Dale, P., Murray, J.L., & Grdina, D.J. (1996). The inhibition of radiation-

induced mutagenesis by the combined effects of selenium and the aminothiol WR-

1065. Mutation Research, Vol. 356, No.2, pp.147-154

Egner, P.A., Wang, J.B., Zhu, Y.R., Zhang, B.C., Wu, Y., Zhang, QN., Qian, G.S., Kuang, S.Y.,

Gange, S.J, Jacobson, L.P., et al. (2001). Chlorophyllin intervention reduces

aflatoxin-DNA adducts in individuals at high risk for liver cancer. Proceeding In

National Academy of Science, U S A, Vol. 98, No.25, pp.14601-14606

Farooqi, Z., & Kesavan, P.C. (1992). Radioprotection by caffeine pre- and post-treatment in

the bone marrow chromosomes of mice given whole-body gamma-irradiation.

Mutation Research, Vol. 269, No.2, pp.225-230

George, K.C., Hebbar, S.A., Kale, S.P., & Kesavan, P.C. (1999). Caffeine protects mice against

whole-body lethal dose of gamma-irradiation. Journal of Radiological Protection,

Vol.19, No.2, pp.171-176

Selected Topics in DNA Repair

494

Glacet-Bernard, A., Coscas, G., Chabanel, A., Zourdani, A., Lelong, F., & Samama, M.M.

(1994). A randomized, double-masked study on the treatment of retinal vein

occlusion with troxerutin. Americal Journal of Ophthalmology, Vol. 118, No.4, pp.421-

429

Guo, D., Horio, D.T., Grove, J.S., & Dashwood, R.H. (1995). Inhibition by chlorophyllin of 2-

amino-3-methylimidazo-[4,5-f]quinoline-induced tumorigenesis in the male F344

rat. Cancer Letters, Vol. 95, No.1-2, pp.161-165

Hebbar, S.A., Mitra, A.K., George, K.C., & Verma, N.C. (2002). Caffeine ameliorates

radiation-induced skin reactions in mice but does not influence tumour radiation

response. Journal of Radiological Protection, Vol. 22, No.1, pp.63-69

Imanishi, H., Sasaki, Y.F., Matsumoto, K., Watanabe ,M., Ohta, T., Shirasu, Y., & Tutikawa,

K. (1990). Suppression of 6-TG-resistant mutations in V79 cells and recessive spot

formations in mice by vanillin. Mutation Research, Vol. 243, No.2, pp.151-158

Jagetia, G.C. (2007). Radioprotective Potential of Plants and Herbs against the Effects of

Ionizing Radiation. Journal of Clinical Biochemistry and Nutrition, Vol. 40, No.2,

pp.74-81

Johnson, S. M., Torrice, C. D., Bell, J. F., Monahan, K. B., Jiang, Q., Wang, Y., Ramsey, M. R.,

Jin, J., Wong, K., Su, L., Zhou, D., & Sharpless, N. E. (2010). Mitigation of

hematologic radiation toxicity in mice through pharmacological quiescence

induced by CDK4/6 inhibition. The Journal of Clinical Investigation, Vol 120, No. 7,

pp.2528–2536.

Kapoor, S., Mukherjee, T., Kagiya, T.V., & Nair, C.K. (2002). Redox reactions of tocopherol

monoglucoside in aqueous solutions: a pulse radiolysis study. Journal of Radiation

Research (Tokyo), Vol. 43, No.1, pp.99-106

Keshava, C., Keshava, N., Ong, T.M., & Nath, J. (1998). Protective effect of vanillin on

radiation-induced micronuclei and chromosomal aberrations in V79 cells. Mutation

Research, Vol. 397, No.2, pp.149-159

Kim, J.K., Kim, J.H., & Yoon, Y.D. (2003). Evaluation of caffeine as a radioprotector in whole-

body irradiated male mice. In Vivo, Vol.17, No.2, pp.197-200

Kumar, S.S., Chaubey, R.C., Devasagayam, T.P.A., Priyadarsini, K.I., & Chauhan, P.S. (1999).

Inhibition of radiation-induced DNA damage in plasmid pBR322 by chlorophyllin

and possible mechanism(s) of action. Mutation Research, Vol. 425, No.1, pp.71-79

Kumar, S.S., Devasagayam, T.P.A., Jayashree, B., & Kesavan, P.C. (2001). Mechanism of

protection against radiation-induced DNA damage in plasmid pBR322 by caffeine.

International Journal of Radiation Biology, Vol. 77, No.5, pp.617-623

Kumar, S.S., Shankar, B., & Sainis, K.B. (2004). Effect of chlorophyllin against oxidative

stress in splenic lymphocytes in vitro and in vivo. Biochim Biophys Acta, Vol.1672,

No.2, pp.100-111

Kuna, P. (1983). Duration and degree of radioprotection of WR-2721 in mice following its

intraperitoneal, intramuscular and subcutaneous administration. Radiobiological

Radiotherapy (Berl), Vol.24, No.3, pp.357-364

Landauer, M.R., Srinivasan, V., & Seed, T.M.. (2003). Genistein treatment protects mice from

ionizing radiation injury. Journal of Applied Toxicology, Vo. 23, No.6, pp.379-385

Lee, E.K., Kim, J.M., Choi, J., Jung, K.J., Kim, D.H., Chung, S.W., Ha, Y.M., Yu, B.P., &

Chung, H.Y. (2011). Modulation of NF-kappaB and FOXOs by baicalein attenuates

the radiation-induced inflammatory process in mouse kidney. Free Radicical

Research, Vol. 45, No.5, pp.507-517

Lee, J.H., & Park, J.W. (2004). A manganese porphyrin complex is a novel radiation

protector. Free Radical Biology in Medicine, Vol. 37, No.2, pp.272-283

Role of Radioprotectors in the Inhibition of DNA Damage

and Modulation of DNA Repair After Exposure to Gamma-Radiation

495

Lefebvre, G., & Lacombe, C. (1991). Venous insufficiency in the pregnant woman.

Rheological correction by troxerutin. Rev Fr Gynecol Obstet, Vol.86(2 Pt 2), pp.206-

208

Ma, Z.C., Hong, Q., Wang, Y.G., Tan, H.L., Xiao, C.R., Liang, Q.D., Lu, B.B., & Gao, Y.

(2011a). Effects of ferulic acid on hematopoietic cell recovery in whole-body gamma

irradiated mice. International Journal of Radiation Biology.

(doi:10.3109/09553002.2011.548438)

Ma, Z.C., Hong, Q., Wang, Y.G., Tan, H.L., Xiao, C.R., Liang, Q.D., Wang, D.G., & Gao, Y.

(2011b). Ferulic acid protects lymphocytes from radiation-predisposed oxidative

stress through extracellular regulated kinase. International Journal of Radiation

Biology, Vol.87, No.2, pp.130-140

Maurya, D.K., Adhikari, S., Nair, C.K.K., & Devasagayam T.P.A. (2007). DNA protective

properties of vanillin against gamma-radiation under different conditions: possible

mechanisms. Mutation Research, Vol. 634, No.1-2, pp.69-80

Maurya, D.K., Balakrishnan, S., Salvi, V.P., & Nair, C.K.K. (2005a). Protection of cellular

DNA from gamma-radiation-induced damages and enhancement in DNA repair by

troxerutin. Molecular and Cellular Biochemistry, Vol. 280, No.1-2, pp.57-68

Maurya, D.K., Devasagayam, T.P.A., & Nair, C.K.K. (2006). Some novel approaches for

radioprotection and the beneficial effect of natural products. Indian Journal of

Experimental Biology, Vol. 44, No.2, pp.93-114

Maurya, D.K., & Nair, C.K.K. (2006). Preferential radioprotection to DNA of normal tissues

by ferulic acid under ex vivo and in vivo conditions in tumor bearing mice.

Molecular and Cellular Biochemistry, Vol. 285, No.1-2, pp.181-190

Maurya, D.K., Salvi, V.P., & Nair, C.K.K. (2004). Radioprotection of normal tissues in tumor-

bearing mice by troxerutin. Journal of Radiation Research (Tokyo), Vol. 45, No.2,

pp.221-228

Maurya, D.K., Salvi, V.P., & Nair, C.K. K.(2005b). Radiation protection of DNA by ferulic

acid under in vitro and in vivo conditions. Molecular and Cellular Biochemistry, Vol.

280, No.1-2, pp.209-217

Monobe, M., Hino, M., Sumi, M., Uzawa, A., Hirayama, R., Ando, K., & Kojima, S. (2005).

Protective effects of melatonin on gamma-ray induced intestinal damage.

International Journal of Radiation Biology, Vo. 81, No.11, pp.855-860

Moulder, J.E., & Cohen, E.P. (2007). Future strategies for mitigation and treatment of chronic

radiation-induced normal tissue injury. Seminal in Radiation Oncology, Vol. 17, No.2,

pp.141-148

Nair, C.K.K., Parida, D.K., & Nomura, T. (2001). Radioprotectors in radiotherapy. Journal of

Radiatin Research (Tokyo), Vol. 42, No.1, pp.21-37

Nair, C.K.K., Salvi, V., Kagiya, T.V., & Rajagopalan, R. (2004). Relevance of radioprotectors

in radiotherapy: studies with tocopherol monoglucoside. Journal of Environmental

Pathology, Toxicology and Oncology, Vol 23, No.2, pp.153-160

Nair, C.K.K., Devi, P.U., Shimanskaya, R., Kunugita, N., Murase, H., Gu, Y-H., & Kagiya,

T.V. (2003). Water soluble vitamin E (TMG) as a radioprotector. Indian Journal of

Experimental Biology, Vol.41, pp.1365-1371

Nayak, V., & Devi, P.U.( 2005). Protection of mouse bone marrow against radiation-induced

chromosome damage and stem cell death by the ocimum flavonoids orientin and

vicenin. Radiation Research, Vol. 163, No.2, pp.165-171

Rajagopalan, R., Wani, K., Huilgol, N.G., Kagiya, T.V., & Nair, C.K.K. (2002). Inhibition of

gamma-radiation induced DNA damage in plasmid pBR322 by TMG, a water-

Selected Topics in DNA Repair

496

soluble derivative of vitamin E. Journal of Radiation Research (Tokyo), Vol. 43, No.2,

pp.153-159

Roots, R., Kraft, G., & Gosschalk, E. (1985). The formation of radiation-induced DNA breaks:

the ratio of double-strand breaks to single-strand breaks. International Journal of

Radiation Oncology Biology and Physics, Vol. 11, No.2, pp.259-265

Salvi, V.P., Maurya, D.K., Kagiya, T.V., & Nair, C.K.K. (2007). Enhancement in repair of

radiation-induced DNA strand breaks in vivo by tocopherol monoglucoside.

International Journal of Low Radiation, Vol.4, No.1, pp.43-52

Sasaki, Y.F., Ohta, T., Imanishi, H., Watanabe, M., Matsumoto, K., Kato, T., & Shirasu, Y.

(1990). Suppressing effects of vanillin, cinnamaldehyde, and anisaldehyde on

chromosome aberrations induced by X-rays in mice. Mutation Research, Vol. 243,

No.4, pp.299-302

Satyamitra, M., Devi ,P.U., Murase, H, & Kagiya, V.T. (2001). In vivo radioprotection by

alpha-TMG: preliminary studies. Mutation Research, Vol. 479, No.1-2, pp.53-61

Serin, M., Gulbas, H., Gurses, I., Erkal, H.S., & Yucel, N. (2007). The histopathological

evaluation of the effectiveness of melatonin as a protectant against acute lung

injury induced by radiation therapy in a rat model. International Journal of Radiation

Biology, Vol. 83, No.3, pp.187-193

Spielberger, R., Stiff, P., Bensinger, W., Gentile, T., Weisdorf, D., Kewalramani, T., Shea, T.,

Yanovich, S., Hansen, K., Noga, S., et al. (2004). Palifermin for oral mucositis after

intensive therapy for hematologic cancers. New England Journal of Medicine, Vol.

351, No.25, pp.2590-2598

Spotheim-Maurizot, M., & Davidkova, M. (2011). Radiation damage to DNA in DNA-

protein complexes. Journal of Physics: Conference Series 261 (2011)

(doi:10.1088/1742-6596/261/1/012010)

Spothem-Maurizot, M., Mostafavi, M., Douki, T., & Belloni, J., editors. (2008). Radiation

Chemistry: from basics to applications in material and life science: EDP Science.

Stone, H.B., Moulder, J.E., Coleman, C.N., Ang, K.K., Anscher, M.S., Barcellos-Hoff, M.H.,

Dynan, W.S., Fike, J.R., Grdina, D.J., Greenberger, J.S., et al. (2004). Models for

evaluating agents intended for the prophylaxis, mitigation and treatment of

radiation injuries. Report of an NCI Workshop, December 3-4, 2003. Radiation

Research,Vol. 162, No.6, pp.711-728

Tilak, J.C., & Devasagayam, T.P.A. (2003). Radioprotective property of baicalein. BARC

Newsletter, Vol. 249, pp.98–104

Ueno, M., Inano. H., Onoda, M., Murase, H., Ikota, N., Kagiya, T.V., & Anzai, K. (2009).

Modification of mortality and tumorigenesis by tocopherol-mono-glucoside (TMG)

administered after X irradiation in mice and rats. Radiation Research, Vol. 172, No.4,

pp.519-524

Devi, P.U., Ganasoundari, A., Vrinda, B., Srinivasan, K.K., & Unnikrishnan, M.K. (2000).

Radiation protection by the ocimum flavonoids orientin and vicenin: mechanisms

of action. Radiation Research, Vol 154, No.4, pp.455-460

Vijayalaxmi, Meltz, M.L., Reiter, R.J., Herman, T.S., & Kumar, K.S. (1999). Melatonin and

protection from whole-body irradiation: survival studies in mice. Mutation

Research, Vol. 425, No.1, pp.21-27

Vijayalaxmi, Reiter, R.J., Herman, T.S., & Meltz, M.L. (1996). Melatonin and radioprotection

from genetic damage: in vivo/in vitro studies with human volunteers. Mutation

Research, Vol. 371, No.3-4, pp.221-228

Weiss, J.F., & Landauer, M.R. (2009). History and development of radiation-protective

agents. International Journal of Radiation Biology, Vo. 85, No.7, pp.539-573

DNA-Binding Radioprotectors

Pavel Lobachevsky

, Alesia Ivashkevich

Olga A. Martin

1,2

and Roger F. Martin

Laboratory of Molecular Radiation Biology,

Peter MacCallum Cancer Centre

Laboratory of Molecular Pharmacology,

Center for Cancer Research, National Cancer Institute

Australia

USA

1. Introduction

For decades the world of radioprotectors has been dominated by the aminothiols, in

particular WR1065 and its prodrug amifostine. These drugs emerged from an extensive

programme of synthesis and evaluation under the auspices of the Walter Reed Army

Institute of Research starting in the early 1950s (Sweeny, 1979). As discussed in detail in

section 4 below, structure-activity studies on a series of aminothiols in John Ward’s lab at

the University of San Diego established a relationship between net charge and

radioprotective activity. Positive charge conferred a DNA binding capability, by ionic

interaction, and improved radioprotective activity. This was consistent with the fact that an

important aspect of the mechanism of radioprotection by WR1065 is its radical scavenging

activity. Given the limited range of diffusion of hydroxyl radicals generated from ionisation

of water molecules, it makes sense that the radical scavengers will be most effective when

located in the close vicinity of DNA. This basic rationale prompted the synthesis and

evaluation of an aminothiol tethered to a DNA intercalating agent (Laayoun et al., 1994), but

there is no evidence in the literature of a systematic follow-up.

Also, the new DNA binding radioprotector methylproamine emerged not from a rational

design premise, but rather, from the serendipitous discovery of radioprotective activity of a

minor groove binder Hoechst 33342 synthesized by the Hoechst company as part of a

program aimed at developing antihelminthics. From that starting point, a modest lead

optimisation program guided by a mechanistic hypothesis showed that radioprotective

activity was enhanced by the introduction of more electron-rich substituents into the phenyl

ring of the molecule.

Thus, this article links two groups of radioprotectors with the common feature of DNA-

binding, albeit with quite different affinities. The dissociation constant for the WR1065-DNA

interaction is in the mM range (Smoluk et al., 1986), whereas that for methylproamine is a

few hundred nM (Martin et al., 2004). Accordingly, the relative radioprotective potency of

WR1065 and methylproamine differs by more than 2-orders of magnitude. In contrast to this

focus, other publications review a much wider range of radioprotectors (Hosseinimehr,

2007; Weiss & Landauer, 2009; Citrin, 2010).

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2. DNA damage and biological response to radiation

Both the hazards and potential beneficial uses of ionizing radiation (IR) were realised soon

after the discovery of X-rays by Wilhelm Conrad Roentgen in 1895. Studies of Hiroshima

and Nagasaki survivors, reconstructed dosimetry, and unfortunate accidents at nuclear

plants documented a pattern of events following a whole-body IR exposure, confirmed by

extensive animal experiments. Exposure to high doses of IR (100-150 Gy) leads to death

within a few hours which results from neurological and cardiovascular breakdown. At

intermediate dose levels (5-12 Gy), death occurs within a few days and is associated with

gastrointestinal syndrome. At lower doses (2.5-5 Gy) death occurs within several weeks due

to haematopoietic syndrome (Hall, 1973). All these effects are attributable to killing of

critical cells, and the question of how IR kills cells has stimulated much research. A key

milestone was the identification of DNA as the critical molecular target. This research was

prompted by both the potential uses of IR, for example in cancer therapy, and by concerns

about effects of IR on health. Potentially damaging exposure may come from diagnostic

radiology such as computed tomography as well as from cosmic rays, the sun and

radioactive nuclides in the ground (e.g. radon), during high altitude journeys, or in space.

Such concerns about occupational and environmental radiation exposure have prompted

much scientific and legislative activity, the latter leading to the establishment of the

International Commission on Radiological Protection.

It is a commonly recognised concept that two distinct mechanisms are responsible for

induction of DNA damage by IR; one involves direct ionisation of atoms in the DNA

molecule and usually is referred to as the direct effect, and another that results from DNA

attack by free radicals generated as a result of the radiolysis of surrounding water molecules

and is referred to as the indirect effect (Hall, 1973; Hall et al., 1988). The major contributor to

the indirect effect is the hydroxyl radical (HO•) as evidenced by studies using compounds

that scavenge hydroxyl radicals (Roots & Okada, 1972). The direct DNA damage is

considered to include the damage that is produced by hydroxyl radicals generated in water

molecules intimately associated with DNA water layer since these radicals cannot be

scavenged (Ward, 1994a). It is estimated that two-thirds of DNA damage is caused

indirectly by scavengeable radicals (Roots & Okada, 1972).

IR induces a wide variety of mainly isolated DNA lesions, including strand breaks (single

strand breaks – SSB) and damage that involves DNA bases (modified bases and abasic sites).

Isolated DNA lesions are normally easily repaired by cells (Ward, 1994a; Sutherland et al.,

2000), without serious biological consequences. It is believed that cytotoxic and mutagenic

effects of IR originate from so called clustered DNA damage when two or more lesions

occur close to each other in both DNA strands (Goodhead, 1994; Ward, 1994a; Goodhead &

Nikjoo, 1997; Nikjoo et al., 1997; Nikjoo et al., 1998; Sutherland et al., 2000). This type of

DNA damage was initially termed locally multiply damaged sites (LMDS) (Ward, 1994a),

but more recently the term LDMS has been largely replaced by the synonym oxidative

clustered DNA lesions (OCDL) (Sutherland et al., 2000; Georgakilas, 2008). Formation of

strand breaks in opposite DNA strands results in a double strand break (DSB), that

represents a particular case of OCDL (Purkayastha et al., 2007). Assuming that base damage

(BD) occurs more frequently than SSB (Ward, 1994b; Ward, 1994a), it is expected that the

majority of OCDL are not frank DSB but contain two BD or BD and SSB in opposite strands

(Goodhead & Nikjoo, 1997). While isolated DNA lesions are generally repaired efficiently,

OCDL are more difficult to resolve (Ward, 1981; Harrison et al., 1999; Georgakilas et al.,

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2002; Georgakilas, 2008). An attempt to process by the base excision repair (BER) a modified

base that constitute an OCDL with a SSB in opposite strand may result in formation of a

DSB, which also can be formed when an unprocessed OCDL interferes with either DNA

replication and transcription (Bonner et al., 2008; Sedelnikova et al., 2010). Although DNA

DSB can be repaired in cells by non-homologous end joining (NHEJ) and homologous

recombination (HR) (Matsumoto et al., 1994; Memisoglu & Samson, 2000; Wilson et al., 2003;

Cadet et al., 2010; Hinz, 2010; Lieber, 2010; Mladenov & Iliakis, 2011), DSB are among the

most toxic IR-induced DNA lesions. If not properly repaired, both accumulated DSB and

OCDL lead to cytotoxicity, genome instability and carcinogenesis (Jeggo & Lobrich, 2007;

McKinnon & Caldecott, 2007).

It is established that a substantial level of oxidative DNA lesions may be present in normal

cells and tissues, usually a few isolated oxidative DNA lesions per Mbp (Nakamura &

Swenberg, 1999; De Bont & van Larebeke, 2004). These lesions are believed to be generated

by free radicals that originate from endogenous reactive oxygen species (ROS) (Riley, 1994;

Mikkelsen & Wardman, 2003). Two of the biologically important endogenous ROS are

superoxide anion radical O

• and hydrogen peroxide H

. Endogenous superoxide is

produced in cells mainly as a result of mitochondrial respiration (Mikkelsen & Wardman,

2003), and then is efficiently converted to hydrogen peroxide by cellular superoxide

dismutase (SOD). Although the superoxide and hydrogen peroxide are relatively long lived

species and are able to diffuse in cells over considerable distance (Riley, 1994), these

endogenous species are produced in cytoplasm and also they are not able to damage DNA

directly. The genotoxic effect of the endogenous ROS is mainly mediated by their ability to

give rise to hydroxyl radicals from hydrogen peroxide by a redox reaction with traces of

reduced transitional metal ions, mainly ferrous via Fenton chemistry (Mikkelsen &

Wardman, 2003). Since ROS represent potential risk for cells, an antioxidant defense system

has been developed in cells to maintain a steady state level of ROS.

Exogenous cytotoxic agents can lead to the increase above the steady state in the ROS level

thus creating an oxidative stress that can result in induction of additional oxidative DNA

damage (Sedelnikova et al., 2010). Exposure to IR is also known to cause the oxidative stress

however, interestingly, the level of ROS generated directly from radiolysis of water at

biologically relevant doses is much less that the level of the endogenous ROS. This follows

from the estimation that for example a 100 Gy radiation dose would be required to double

the endogenous level of one of the major types of DNA base damage 7,8-dihydro-8-

oxoguanine (8-oxoG) (Ward, 1994b). There is also experimental evidence that the transient

increase in the cellular ROS level following irradiation is dependent on mitochondria

respiration, however it is dose independent in the range of biologically relevant doses (1 –

10 Gy) with the fraction of cells exhibiting the increased ROS level being dose dependent

(Leach et al., 2001). These observations underline the minimal impact of isolated DNA

lesions and the critical role of clustered lesions for cyto- and genotoxic consequences of IR.

The major difference between endogenous ROS and those generated by IR is that while the

spatial distribution of hydroxyl radicals produced from endogenous ROS is random, IR is

also able to generate clusters of hydroxyl radicals within a nanometre scale resulting in the

multiple radical attack on DNA within small volume from a single track of a charged

particle (Goodhead, 1994; Goodhead & Nikjoo, 1997; Nikjoo et al., 1997; Nikjoo et al., 1998).

As a result, the relative frequency of OCDL is much higher for damage induced by IR as

compared to endogenous oxidative DNA damage for which OCDL are very rare. The

frequency of endogenous OCDL is estimated to be a few per Gbp in normal tissues (Bennett

Selected Topics in DNA Repair

500

et al., 2005), and can probably be formed as a result of two isolated oxidative lesions

occurring close to each other spatially and temporally. The radiation induced OCDL can

also be more complex since consisting of more than two individual lesions in a cluster

(Ward, 1994a; Bennett et al., 2005).

Recently, the dogma that cells subjected to IR are killed solely through direct energy

deposition within a cell, with the effect being proportional to dose, has been reconsidered in

view of the discovery of the radiation-induced bystander effect (RIBE). The neighbours of

irradiated cells respond as if they themselves have been irradiated. The RIBE is now a well-

established consequence of exposure to IR and is manifested as increased genomic

abnormalities and loss of viability in unirradiated (“bystander”) cells associated with the

targeted cells. Affected bystander cells exhibit increased levels of micronuclei, apoptosis,

mutations, altered DNA damage and repair, and senescence arrest (Sokolov et al., 2005;

Sedelnikova et al., 2007; Prise & O'Sullivan, 2009). DSB appear in the DNA of bystander

cells, invoking the existence of some kind of biological “danger signal” that is sent from

irradiated to bystander cell. Possible mediators of the RIBE include various inflammation-

related cytokines and ROS including nitric oxide that have been found at elevated levels in

medium conditioned by irradiated cells (Dickey et al., 2009; Prise & O'Sullivan, 2009; Hei et

al., 2010; Ivanov et al., 2010).

3. Radiation therapy of tumours and the role of radioprotectors

The detrimental consequences of IR for cells and tissues can be harnessed in cancer radiation

therapy. Radiation therapy exploits the cytotoxic effect of IR on cancer cells (Lawrence et al.,

2008) and represents one of the three major treatment modalities for cancer, along with

surgery and cytotoxic chemotherapy. Radiotherapy is used in approximately half of all

patients diagnosed with cancer at some stage of their illness. Technological advances in the

physical targeting of radiation to the tumour are extensively exploited, reflecting the simple

idea that the most efficient radioprotection strategy is to exclude normal tissues from

irradiated volume. These include such techniques as conformal radiotherapy, intensity

modulated therapy, image guided radiotherapy etc (Brizel, 2005). Although still in the pre-

clinical stage of development, microbeam radiotherapy (MRT), in which the X-ray beam is

split into an array of planar parallel microbeams, shows much greater therapeutic index

than conventional radiotherapy. Studies of synchrotron MRT in animal models indicated

that tumours can be ablated by MRT at radiation levels that spare normal tissues (Dilmanian

et al., 2002; Dilmanian et al., 2003; Crosbie et al., 2010). Nevertheless, whether used alone or

in combination with other treatment modalities, the dose of radiation that can be safely

delivered is limited by radiation induced injury to the normal tissues in the irradiated

volume. This gives rise to the concept of “treatment to tolerance” i.e. the administration of

the maximally tolerated dose (MTD) imposed by the normal tissues which is often less than

required to effect a high probability of tumour eradication. Clearly, any strategy that

selectively increases the MTD improves the chances of tumour cure, and one of such

strategies is the selective pharmacologic modification of the normal tissue response with

radiation modifiers/protectors. These agents alter the response of normal tissues to

irradiation when present in tissues prior or after exposure to IR (Citrin, 2010). This approach

can also be viewed as an attractive countermeasure for possible nuclear/radiological

terrorism and radiation accidents, but without the important constraint to avoid protection

of the tumour that is imperative in the use of radioprotectors in radiation oncology.